Merocyanine 540 (MC540)-mediated photodynamic action is a novel approach for purging
tumor cells from autologous remission bone marrow explants. The purpose of this study was to evaluate the effects of
hemin (
ferriprotoporphyrin IX), a potential source of
pro-oxidant iron in bone marrow, on in vitro photodynamic inactivation of
leukemia cells. Murine L1210 cells exhibited a progressive loss of clonogenicity when irradiated with broad-band visible light in the presence of MC540.
Hemin had strikingly different effects on photokilling, depending on its contact time with cells, eliciting a sizable decrease in resistance after short-term (30-min) contact but a marked increase in resistance after long-term (24-h) contact. Similar trends were observed when cells were challenged with
glucose/
glucose oxidase, indicating that the responses apply to more than one type of oxidative stress. Immunoblot analyses revealed that the levels of inducible
heme oxygenase (HO-1) and
ferritin heavy (H) chain were substantially elevated 24 h after
hemin addition. HO-1 increased relatively rapidly and maximized within 4 h after adding
hemin, whereas
H-ferritin increased more slowly in parallel with the development of hyperresistance, maximizing after 24-36 h.
Desferrioxamine, an avid
iron chelator, had no effect on HO-1 induction but inhibited both
ferritin induction and the increase in cell resistance, suggesting that HO-mediated release of
iron from
hemin was necessary for triggering these responses. Spleen
apoferritin was taken up by L1210 cells and strongly inhibited photokilling, further implicating
ferritin involvement in hyperresistance. Photokilling was accompanied by
free radical-mediated lipid peroxidation (
thiobarbituric acid reactivity), which could be suppressed substantially by 24-h
hemin preincubation. A plausible explanation for the long-term effects of
hemin is that excess
H-ferritin generated as a result of
iron-regulatory protein deactivation sequesters toxic
iron, which might otherwise catalyze damaging lipid peroxidation. Chronic oxidative release of
hemin from bone marrow erythroid cells could compromise the efficacy of photopurging by making
tumor cells more tolerant to photooxidative insult.